DETECTION OF THE T(2-13)(Q35-Q14) AND PAX3-FKHR FUSION IN ALVEOLAR RHABDOMYOSARCOMA BY FLUORESCENCE IN-SITU HYBRIDIZATION

Cytogenetic studies of the pediatric solid tumor alveolar rhabdomyosar coma have demonstrated the presence of a consistent chromosomal transl ocation, t(2;13)(q35;q14). We recently identified PAX3 and FKHR as the genes on chromosomes 2 and 13, respectively, that are juxtaposed by t his translocation. As one means of detecting the t(2; 13) translocatio n in clinical specimens, we have developed a fluorescence in situ hybr idization (FISH) assay that may be used for both interphase and metaph ase cells. Translocation of the 5' region of the FKHR gene to the deri vative chromosome 2, and retention of the 3' region of FKHR on the der ivative chromosome 13[(der(13)], were demonstrated in metaphase cells from a rhabdomyosarcoma cell line with a previously identified t(2;13) translocation. A 5' PAW cosmid probe was shown to localize to 2q35 in normal cells, and to translocate to the der(13) in the rhabdomyosarco ma cell line. In order to detect the der(13) in interphase nuclei, we labeled the 3'FKHR and the 5'PAX3 cosmid probes with digoxigenin and b iotin, respectively, and used these in a two-color FISH assay. The pre sence of the der(13) was visualized as juxtaposed or overlapping red a nd green signals in metaphase and interphase tumor cells. The PAX3-FKH R FISH assay was then applied to a series of cytogenetically character ized pediatric sarcoma cell lines. The presence of the der(13) was dem onstrated by FISH in all cases containing a cytogenetically detectable t(2;13). The FISH assay was then applied to a series of 20 embryonal and alveolar rhabdomyosarcoma samples. All 10 of the alveolar rhabdomy osarcoma specimens demonstrated a der(13) with the FISH assay. We did not detect a PAX3-FKHR fusion in 10 embryonal rhabdomyosarcoma cases. Thus, the two-color FISH assay is a sensitive and rapid means of ident ifying the t(2;13) in rhabdomyosarcoma specimens, and it will be a use ful adjunct for the diagnosis of pediatric small round cell rumors. Th e cosmid probes for the 5' and 3' regions of FKHR, as well as the prob e for PAX3, will be useful for molecular cytogenetic studies of varian t translocations in rhabdomyosarcoma, such as the t(1;13)(p36;q14). (C ) 1995 Wiley-Liss, Inc.